Tuesday, April 5, 2011

Multi-Color FACS

As you may remember, we are using the Flow Cytometer (FACS) to measure the fluorescence levels of individual cells. Our FACS is an older BD FACSCalibur that has gone through several updates. Almost exactly a year ago, we had all the electronic boards refurbished. At the same time we already had plans to replace the old 488nm laser with two lasers at different colors. These lasers arrived with the electronics, but we decided to hold off on their installation that until we stop using GFP markers (that need 488nm excitation). 

After few months we realized that this will not happen so fast, and ordered a third laser with 488nm capabilities. Due to many reasons the delivery of this upgrade was postponed for a long while. Finally, last week, all the necessary parts arrived. Last week Ariel and David from Merkel Technologies installed these upgrades and today we had a training session to go over the capabilities of these devices.

As part of the installation, Ariel removed the old (big and heat generating) laser, and put in a stack of three lasers instead. These required a panel of one-way mirrors to focus the beams onto the light path of the flowcytometer.

You can see the lasers here (with Ariel explaining) on the left side as small boxes. They are really tiny, but sit on aluminum blocks to disperse heat they generate.

The new light path involves mirrors that take the beams from the lasers

More schematically, the new light path configuration is shown below, where each line segment is either a bandpass filter (that allows only small "window" of wavelengths to pass through) or a half-mirror (that reflect certain wavelengths and passes others). As a result each sensor (FL) measures different area of the spectrum.

The consequence of this design is that we now can measure either GFP and mCherry  (a type of RFP) together, or YFP and mCherry. We cannot operate measure together both GFP and YFP since the YFP excitation is very close to the GFP emission.

As before, when the FACS is serviced all the covers come off, and it is a great chance to take pictures of the light beams going through the prisms and mirrors


and finally hitting the target area in the flow cell

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